Numerous experiments have shown that pregnancy induces a protective effect against tumor development in the mammary glands of both rodents and humans. This protective effect can be mimicked in rats by administration of estrogen and progesterone prior to chemical treatment. Several questions regarding athe hormonal induction of the protective effect on mammary tumorigenesis remain unanswered. These questions involve the dose and duration of hormones required to induce a protective state, the stability of the protective state. This project addresses three specific aims which are relevant to understanding the cellular and molecular basis of the protective effect. The model chosen to address these aims is N-methyl-N- nitrosourea (MNU) induced mammary tumorigenesis in inbred Wistar-Furth rats pretreated with estrogen and progesterone. Specific aim 1 determines the minimum hormone doses and time required to induce a protective effect. The expression of TGFbeta II will be assessed in the resist ant and susceptible mammary gland to provide an endpoint for the resistant cell phenotype. Specific Aim 2 examines the persistence of the resistant state fy administering MNU to rats at 28, 78 and 128 days after the end of hormone treatment. Specific Aim 3 examines whether resistance resides within the mammary gland parenchyma or stromal compartment using the mammary fat pad transplantation system. Glands from both virgin and hormone-treated animals will be transplanted into rats and examined for their respective susceptibility to MNU. Specific Aim 4 will establish mammary epithelial cell lines from susceptible and resistant mammary glands and from MNU-induced mammary tumors. These cell lines will be used as recipient cells for transfection experiments examining the consequences of overexpression of specific genes isolated in other projects. The results of these experiments will establish a minimal hormonal regiment for prevention of experimental breast cancer and a firm rationale for developing hormonal intervention as a feasible approach for human cancer prevention.